Drug screening and diagnosis based on paracrine tubular renin-angiotensin system

ABSTRACT

The present invention relates to a method for screening drugs for use in treating hypertension using the tubular renin-angiotensinogen system identified by the present invention. The invention further relates to a method to diagnose sodium status and sensitivity in an individual by measuring urinary angiotensinogen or angiotensin-I.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is related to U.S. provisional patentapplication Ser. No. 60/099,270 filed Sep. 4, 1998, incorporated hereinby reference.

This invention was made with Government support under Grant No. HL45325awarded by the National Institutes of Health, Bethesda, Md. The UnitedStates Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates to a method for screening drugs for use intreating hypertension using the tubular renin-angiotensinogen systemidentified by the present invention. The invention further relates to amethod to diagnose sodium status in an individual by measuring urinaryangiotensinogen, angiotensin-I, des-AI-angiotensinogen or renin.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedlist of references.

The following abbreviations are used herein: A-I-angiotensin-I;A-II-angiotensin-II; ACE-angiotensin converting enzyme;AGT—angiotensinogen gene; ANG or Ang—angiotensinogen protein;-6(A)/-6(G)—promoter polymorphism at position -6; CCD—corticalcollecting duct; CNT—cortical connecting tubule; DCT—distal convolutedtubule; IC—intercalated cells; JGA—juxtaglomerular apparatus;PCR—polymerase chain reaction; RAS—renin-angiotensin system;RT-PCR—reverse transcriptase polymerase chain reaction; and HPLC—highpressure liquid chromatography.

Blood pressure control is intrinsically linked to fluid volume balanceand electrolyte homeostasis. Regulation of plasma volume in response tovariation in dietary sodium (1) is primarily controlled by therenin-angiotensin system (RAS) and its main effector angiotensin-II(A-II); this peptide hormone is released from angiotensinogen (Ang) bytwo cleavage steps involving renin and angiotensin-converting enzyme(ACE) (2).

The short-term effects of A-II are better understood than its long-termeffects. Acute depletion of body fluid volume triggers a vasoconstrictorresponse mediated by the circulating renin-angiotensin system (RAS),involving renin secreted by the juxtaglomerular apparatus (JGA) in thekidney, Ang from liver, and ACE present in the luminal cell membrane ofcapillary endothelium.

Sustained low-dose infusion of A-II leads to progressive, long-termelevation of arterial pressure due to cumulative sodium retentionprimarily mediated by direct intrarenal A-II effects ¹. A-II has beendetected in proximal tubular luminal fluid at high concentrations (3,4). In contrast to plasma renin (36-40 kDa), Ang (61-65 kDa) is notfiltered through the glomerular basement membrane. Detection of abundantangiotensinogen mRNA in proximal tubule epithelium (5-7), stronglysuggests local generation of A-II at this site by an as yet unspecifiedmechanism. Renin mRNA can be detected in proximal tubule only byapplication of the very sensitive technique of RT-PCR (8). ExogenousA-II stimulates the luminal sodium-hydrogen exchanger present in theproximal tubule cells (9, 10) and also stimulates epithelial sodiumchannels and possibly other transporters in the distal segments ofnephron (11-14).

Fundamental questions remain unanswered, however. If intrarenal A-IIdirectly affects sodium reabsorption, where is it generated, and by whatmechanism? How is this mechanism regulated in response to sodium? Atwhat sites does A-II impact on sodium transport along the nephron? Whatis the mechanism for coordinated regulation of sodium uptake in proximaland distal segments of the nephron? Can it allow for a decoupling ofsodium reabsorption and potassium excretion in the distal tubule?

It is desired to address these questions and to elucidate answers whichcan be used for screening drugs and diagnosing sodium status of anindividual.

SUMMARY OF THE INVENTION

In accordance with the present invention, it is shown that proximaltubule cells cultured as a polarized monolayer secrete Ang at theirapical side, and that Ang transits through the entire nephron as it canbe measured in final urine. Furthermore, it is shown that renin, inaddition to being filtered, is expressed in a specific segment of thenephron, the connecting tubule. Furthermore, angiotensinogen expressionin proximal tubules and renin expression in connecting segments (distalarcades) is an inverse function of dietary sodium.

The data disclosed herein suggest that filtered renin, Ang secreted intoproximal tubule, and renin in connecting tubule, together with luminalangiotensin-converting enzyme (ACE) and A-II receptors, previouslydemonstrated in the luminal fluid and the apical cellular membranes ofproximal tubules and collecting ducts (15, 16), define a tubular RASinvolved in the control of sodium reabsorption as a function of dietarysalt. This tubular RAS could contribute to body fluid control and bloodpressure regulation. Furthermore, genetic differences in theangiotensinogen gene (17, 18) may influence susceptibility to essentialhypertension through their impact on this tubular system.

Thus, the present invention relates to a method for screening drugs foruse in treating hypertension using the tubular renin-angiotensinogensystem identified by the present invention. The invention furtherrelates to a method to diagnosis sodium status in an individual bymeasuring urinary angiotensinogen, angiotensin-I, des-AI-angiotensinogenor renin.

It has been discovered that angiotensinogen, its enzyme catalyzedproducts or renin excreted in urine vary with changes in dietary sodium.Thus, the sodium status of an individual is diagnosed by determining theamount of angiotensinogen or its enzyme catalyzed products or renin inthe urine of the individual and comparing the determined amount withnormal values. A finding of elevated levels of these compounds indicateshigh sodium. An individual's sodium sensitivity can also be determinedby determining the amounts of these compounds in urine. Levels of thesecompounds are determined using conventional techniques, and anyappropriate method is suitable for use. If the levels are elevated in anindividual under a high salt diet compared to reference values, theindividual is sensitive to salt.

It has also been found that the expression of angiotensinogen and reninis regulated at specific sites along the kidney. This finding identifiesnew therapeutic targets for the blood pressure control and provides thebasis for a method to screen drugs for use in treating hypertension.According to the present invention, drug candidates for treatinghypertension are screened using the tubular renin-angiotensin system bytesting the effects of drug candidates at the proximal and/or distaltubule.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIGS. 1A, 1B, 1C and 1D show synthesis and secretion of angiotensinogen(Ang) in the nephron. FIG. 1A shows localization of angiotensinogen inthe nephron of sodium-restricted mice. Immunostaining forangiotensinogen is observed only in proximal tubule cells, identified byPAS counterstaining. High magnification reveals an apical vesicularstaining of Ang under the brush border, suggesting a secretory process.FIG. 1B shows directional secretion of Ang by proximal tubularepithelium in vitro. Western blot analysis using a polyclonal Angantibody demonstrates that proximal tubule cells secrete angiotensinogenexclusively at their apical side. FIG. 1C shows angiotensinogensecretion, measured as urinary Ang (black bars) and urinaryangiotensin-I (A-I; white bars) is an inverse function of sodium status.Ang was measured as the amount of A-I released in a cleavage reaction byrenin; A-I was measured by RIA (means±S.E.M. of three replicates). FIG.1D shows uncleaved native angiotensinogen was also detected in the urineof six healthy male human subjects.

FIGS. 2A-2G show immunolocalization of renin in connecting tubule (CNT)cells. In FIGS. 2A and 2B, tubular renin staining (arrowheads) islocalized to arcades of connecting tubules of midcortical and deepnephrons. Arcades are located in the midcortical labyrinth (L) inbetween the medullary rays (MR) and in the vicinity of radial veins (*).Renin staining is also observed in JGA (arrow). FIG. 2C is a close-upview of CNT cells showing apical renin staining. In FIG. 2D, specificityof renin staining is demonstrated by adsorption of the renin antibodywith purified mouse renin, eliminating all renin immunostaining. FIG. 2Eshows that, in humans, immunoreactive rerin is also present in cells ofthe early cortical collecting duct. In FIGS. 2F and 2G, reninimmunostaining is restricted to principal CNT cells. Serial sectionswere stained for renin (FIG. 2F) or for H′ATPase (FIG. 2G), a marker ofintercalated cells. Renin (arrow) was not expressed in H-ATPase positivecells (arrowhead); staining was mutually exclusive (scale bar A, C=128μm; B, D-F=20 μm).

FIGS. 3A-3E demonstrate de novo renin synthesis in the connecting tubule(CNT). Graphic reconstruction of a microdissected nephron is shown inFIG. 3A, with close-ups of a proximal convoluted tubule (FIG. 3B),glomerulus and macula densa (FIG. 3C), and midcortical CNT arcade (FIG.3D). FIG. 3E shows renin transcription detected by RT-PCR inmicrodissected CNT arcades. Under normal sodium diet, reninamplification products were observed unambiguously in total kidney RNA,RNA from glomeruli (glomer) and RNA from CNT arcades. Insodium-restricted animals, specific signal was strong in glomeruli andCNT arcades and faint in proximal tubules (PCT). GAPDH served as aninternal control.

FIGS. 3F-3K demonstrate de novo renin synthesis in the connecting tubule(CNT), showing localization of renin expression in CNT cells by in situRT-PCT. These figures are representative of four independent replicateexperiments. FIG. 3F shows a positive control section without DNasetreatment, showing uniform perinuclear staining. In FIG. 3G, genomicamplification products were detected in cells of all kidney segments,including glomerulus (*), proximal tubule (arrowhead), and distalnephron segments (arrow). In FIG. 3H, negative control sections, DNasetreated and with specific primers but without reverse transcription,show no renin amplification product. FIGS. 3I-3K show DNase-treated andreverse transcribed sections, revealing specific renin staining in CNTcells (arrow), but not in proximal tubule (arrowhead) or other tubularsegments. In FIG. 3J, high magnification reveals positive staining in asubset of CNT cells (arrow) but not in the proximal tubule (arrowhead).In FIG. 3K, renin mRNA was also detected in juxtaglomerular smoothmuscle cells (arrow). All sections were counterstained with PAS (A-C,scale bar=64 μm; D-F, bar=20 μm; F-H, scale bar=64 μm; I-K, bar=20 μm).

FIGS. 3L-3M demonstrate de novo renin synthesis in the connecting tubule(CNT), showing renin secretion by CNT cells. In FIG. 3L, a subpopulationof CNT cells from microdissected CNT arcades (arrowheads) showspericellular halos of immunoreactive renin; non-renin producing cellsare indicated with asterisks. In FIG. 3M, CHO cells expressing reninserved as positive control. In FIG. 3N, CHO cells expressingangiotensinogen served as negative control.

FIGS. 4A-4E show variation in renin expression in CNT cells ofmidcortical and deep nephrons as a function of sodium load and distalsodium delivery. In FIG. 4A, renin staining (arrows) of afferentarterioles and CNT is minimal following sodium loading. In FIG. 4B, onlyCNT renin staining increased following sodium loading in combinationwith amiloride administration. FIG. 4C shows that sodium restrictionalso significantly increased renin staining in CNT cells (scale bars=64μm). Quantitative immunohistochemistry of renin expression in CNT cells(FIG. 4D) and renin expression in JGA cells (FIG. 4E) bysemiquantitative RT-PCR further confirm these observation (mean±S.E.M.of four independent sets of experiments).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for screening drugs for use intreating hypertension using the tubular renin-angiotensinogen systemidentified by the present invention. The invention further relates to amethod to diagnose sodium status in an individual by measuring urinaryangiotensinogen, angiotensin-I, des-AI-angiotensinogen or renin.

Definition of Sodium Sensitivity

Epidemiology, physiology, pathology and drug response indicate thatessential hypertension encompasses a variety of conditions of unknowncause that cannot be resolved on clinical grounds alone. An importantphysiological distinction is whether or not sodium salt plays asignificant contribution to disease. A dominant hypothesis is that thereare innate differences in individual response to excess dietary sodium,and this factor would account for a significant proportion of cases ofessential hypertension. This class can be defined a “sodium sensitive”hypertension simply to stress the role of this contributing factor inthe development of high blood pressure.

While the significance of sodium in the epidemiology of essentialhypertension is compelling in the aggregate, the relationship betweensodium consumption and blood pressure has been difficult to establish atthe individual level. A variety of protocols have been designed in anattempt to identify individuals who would be particularly vulnerable toexcess sodium consumption. Typical maneuvers include blood pressure orweight response to a standardized sodium load. Another approach has beento monitor change in renal blood flow after infusion of angiotensin-IIin individuals exposed to a high sodium diet, or similar physiologicalmanipulations. In general, these approaches have confirmed that thereare indeed two broad classes of responses to such maneuvers, someresponse or none, leading to the definition of “sodium sensitive” and“sodium resistant” individuals. The overlap between the two groupsremains so large, however, that it precludes the unambiguousidentification of any particular individual as a member of either group.This diagnosis issue has been particularly vexing for medical practice,as the efficacy of either dietary sodium restriction or specifictherapeutic intervention critically depends on the identification ofthis underlying factor.

Sodium sensitivity, then, measures an individual's propensity to respondto excess sodium intake by an increase in either blood pressure orweight. In the context of a chronic condition such ss essentialhypertension, which develops insidiously over decades, and where theadverse consequences of excess sodium intake are reflecting minimal butcumulative attrition over time, it is not altogether surprising thatsuch a differential in chronic response escapes characterization by anacute maneuver. Related concepts will be defined below.

Sodium homeostasis subsumes the overall mechanism by which the bodyregulates the fate of sodium as a function of physiological needs.Sodium balance represents the net difference between intake andexcretion which, on average, is zero. In certain situations, such aspregnancy or after significant blood loss, intake exceeds excretion toaccommodate volume expansion or reexpansion. Sodium status, althoughalmost synonymous with sodium balance, is generally used to characterizedietary sodium status, namely sodium excess, normal sodium, orrestricted sodium intake. Monitoring sodium status is important beforeperforming clinical maneuvers as described above, or more relevant yetto monitor compliance to dietary sodium restriction. If geneticdifferences contribute to sodium sensitivity, then it would be clearlyof clinical relevance to characterize sodium sensitivity as a genetic“liability,” or an innate predisposition to develop high blood pressure.

Significance of Intrarenal A-II in Regulation of Sodium Balance

The link inferred between angiotensinogen and sodium homeostasis resultsfrom its known physiological function. Angiotensin-II (A-II), generatedexclusively from angiotensinogen protein (ANG) through two steps ofenzymatic cleavages catalyzed by renin and angiotensin-converting enzyme(ACE), exerts short-term and long-term effects on vascular tone andblood volume and, as a result, it is a major determinant of bloodpressure. As we argue here, the short-term effects of A-II are betterunderstood than are its long-term effects.

The former reflects the vasoconstrictor effect of A-II at the systemiclevel. Specifically, renin made by a specialized segment of afferentrenal arteries (called the juxtaglomerular apparatus, or JGA) acts inthe general circulation on angiotensinogen released by the liver to formangiotensin-I (A-I), subsequently converted to A-II by ACE in capillaryvessels. Increased circulating A-II would then induce constriction ofarterioles that regulate peripheral vascular resistance and the overallcompliance of the vascular system. When blood volume is depleted, thenet effect of reduced compliance is maintenance of normal bloodpressure. When blood volume is normal, increased vascular resistanceleads to increased arterial pressure.

The characterization of the long-term effects of A-II has proven farmore elusive. Reexpansion of blood volume after depletion requiressodium retention in the kidney (as water “follows” sodium). Under normalconditions, variation in dietary sodium intake leads to compensatoryadjustment of sodium excretion so as to maintain baseline blood pressurewithin narrow limits. A-II has been recognized as the dominant hormonepromoting sodium retention, through both indirect and direct renaleffects.

Indirect effects of A-II are mediated by aldosterone, amineralocorticoid (a steroid affecting mineral metabolism) released bythe adrenal after A-II stimulation. This hormone acts in the distal partof the nephron where it promotes sodium reabsorption and potassiumexcretion. Most textbooks still emphasize the presumed dominance ofaldosterone in promoting sodium retention.

The direct sodium-retaining effects of A-II in the kidney are multipleand varied, affecting both renal hemodynamics, that is, the regulationof blood flow through various parts of the kidney, and the activity ofsodium transporters mediating reabsorption of filtered sodium. Thesedirect effects have been primarily demonstrated by addition of A-II toexperimental preparations, and as such, these experiments have notclarified the actual origin, site of action, and regulation of A-IIaccounting for such effects.

A large body of experimental evidence accumulated over the last twodecades has demonstrated that, in normal physiological states,aldosterone plays only a modest role in regulation of sodium excretionto balance intake. Rather, this function is primarily mediated byintrarenal A-II.

A Paracrine Tubular Renin-Angiotensin System

The essence of the findings described here is as follows:

(1) Angiotensinogen is secreted into tubular fluid by epithelial cellsof the proximal tubule (cells lining the luminal side of this nephronsegment).

(2) AGT expression at this site is a function of sodium status.

(3) Angiotensinogen protein transits through the entire nephron and canbe measured in urine, where it results from proximal tubule secretion(circulating angiotensinogen is not filtered through the glomerularmembrane, by contrast to renin).

(4) Renin is expressed by principal cells of the distal nephron, thevery cells expressing both sodium channel (for sodium reabsorption) anpotassium channel (for potassium secretion under control ofaldosterone).

(5) Renin expression at this site also varies as a function of sodiumstatus.

(6) Blocking the sodium channel of principal cells with amiloride leadsto up-regulation of renin expression in distal nephron, indicating thatsodium translocated by this channel is an important sensing mechanism inthe regulation of renin expression in distal nephron.

(7) A-I and active renin, reflecting activity of distal nephron, can bemeasured in urine.

Proposed Function of This Paracrine System

Massive amounts of sodium are filtered daily, of which 99%, to almost100% are reabsorbed by the kidney (“Sodium Balance”). Differenttransporters are involved in various segments of the nephron (“SodiumTransporters”), and coordination of the activity in each segmentdetermines the final amount of sodium excreted in final urine. It iscommon to contrast the functions of proximal and distal tubule as “bulk”and “fine” sodium reabsorption, respectively. After a bulk phase wherethe majority of filtered sodium, water and various solutes arereabsorbed together by a global process dominated by sodium movement, afine phase allows independent, fine adjustment of each constituent infinal urine. Angiotensinogen expression in proximal tubule suggested itsinvolvement in the bulk phase, but did not provide a mechanism for amore critical role in final adjustment of urinary sodium.

The paracrine system described herein provides an answer. It reveals themechanism by which the renin-angiotensin system regulates sodiumexcretion by integrating function at these two critical sites. Theconcept is novel, and it has extensive implications for diagnosistherapeutic research. The diagnostic implications will be detailed,rather briefly given previous claims and the background given here.

As shown by the examples below, it has been found that proximal tubularepithelium cells synthesize and secrete angiotensinogen, thatangiotensinogen circulates through the entire nephron and can bedetected in urine, that renin is expressed by principal cells of thedistal nephron, and that expression of substrate and enzyme at thesesites is affected by variation in dietary sodium. This previouslyunidentified tubular renin-angiotensin system provides the basis for thedrug screening method of the present invention.

A large number of pharmaceutical drugs have been developed and are usedas antihypertensive agents. They can be classified into broad subclassesas a function of their principle of action and the biochemical functionthey target. As noted above, the renin-angiotensin system (RAS) is offundamental importance in blood pressure control. The most recent drugsdeveloped in the field interfere with this system in one of at leastthree ways. Renin inhibitors are analogs of the angiotensinogen cleavagesite which bind to renin with high affinity, and as such, compete withangiotensinogen. Although effective, these compounds have been oflimited usefulness because of problems in drug delivery. ACE inhibitors,such as captopril or lisinopril, have proven effective and have becomeone of the drugs of choice in the treatment of essential hypertension.The A-II Type 1 receptor inhibitor, Losartan from Dupont-Merck,represents the newest agent developed to counter the physiologicaleffects of A-II. Its high affinity for the major receptor mediating thehemodynamic effects of A-II accounts for its action.

A common feature of these drugs designed to interfere with the RAS isthat they have global, systemic and local effects. Indeed, given thepreeminent role ascribed to the circulating RAS in research precedingthe development of these agents, they reflect the state of knowledge ofthe time at which drug development was developed in these directions.

The results described herein pertaining to the existence and the role ofa paracrine tubular RAS in the regulation of sodium balance suggest newtargets for therapeutic intervention and new methods to screen compoundsand ascertain their biological effectiveness.

Thus, the present invention identifies novel targets for the developmentof antihypertensive agents. The new agents will primarily interfere withthe normal function of the paracrine tubular RAS we describe at eitherthe proximal tubule or in the distal nephron. Compounds can beengineered so as to be delivered at either site and so that theirbiological activity is optimal under the prevailing environment of eachsegment. The net effect is to control sodium reabsorption, and as such,it will prevent the development of essential hypertension in subjectsdeemed sodium sensitive. The drugs may prove most effective in a subsetof hypertensive patients. Together with means to identify such subjects,as we have claimed with AGT the selectivity and specificity of suchdrugs will alleviate the difficulty of choosing a given drug anddetermining effective dosage. It is recognized that any given drug iseffective in only a fraction of patients, and at present, there is nosimple way of predicting if any given patient will respond well to anyparticular agent. Indications may be based on associated manifestations,such as coronary heart disease, but not on actual knowledge about themechanism accounting for hypertension. Both conditions being common,there must be instances where hypertension depends on factors distinctfrom those accounting for coronary disease.

In addition, screening methods are used to determine the efficacy ofcompounds designed so as to interfere with the renin-angiotensin systemat either the proximal or distal tubule. The effects of these compoundscan be monitored at three levels: cellular, tissue and whole organism.In the proximal tubule, cellular response to drugs can be monitored interms of angiotensinogen expression and secretion or in terms of sodiumtransport by the sodium-hydrogen exchanger and other sodium-dependenttransporters. In the distal tubule, targeted drugs will affect theactivity of the sodium channel, the density of A-I receptors, and thesynthesis and release of renin by principal cells. At the tissue level,expression of angiotensinogen and renin can be monitored by any one ofthe methods described herein, including in situ RT-PCR, RT-PCR ofmicrodissected nephron segments, particularly Y-junctions, andimmunohistochemistry. At the level of the entire organism, the efficacyof compounds can be evaluated by measuring parameters of the paracrineRAS in urine, including A-I and A-II total angiotensinogen,des-AI-angiotensinogen, uncleaved angiotensinogen, total renin and reninactivity. Furthermore, the effects of such agents on blood pressure andplasma volume can be monitored.

As shown by the examples below, it has been found that angiotensinogen,its enzyme catalyzed products or renin excreted in urine vary withchanges in dietary sodium. Thus, the sodium status of an individual isdiagnosed by determining the amount of angiotensinogen or its enzymecatalyzed products or renin in the urine of the individual and comparingthe determined amount with normal values. Any method for detectingurinary angiotensinogen or angiotensin-I can be used in accordance withthe present invention. A finding of elevated levels of these compoundsindicates high sodium. The levels of these compounds are determinedusing conventional techniques and any appropriate method is suitable foruse. An individual's sodium sensitivity can also be determined bydetermining the amounts of these compounds in urine. If the levels areelevated in an individual under a high salt diet compared to referencevalue, the individual is sensitive to salt.

It has also been found that expression of angiotensinogen and renin isregulated at specific sites along the kidney. This finding identifiesnew therapeutic targets for blood pressure control and provides a basisfor a method to screen drugs for use in treating hypertension. Accordingto the present invention, drug candidates for treating hypertension arescreened using the tubular renin-angiotensin system by testing theeffects of drug candidates at the proximal and/or distal tubule.

Molecular variants in the angiotensinogen gene (AGT) may reflectindividual predisposition to the development of essential hypertension,as we claimed earlier with t235 and a(−6) variants. The actualmanifestation of the genetic propensity evidently depends on the degreeand the duration of the exposure to high sodium intake as well as otherpromoting factors such as overweight and excess stress.

Angiotensinogen, A-I and active renin can be measured in urine inanimals and humans. Furthermore, the amount of angiotensinogen detectedin urine reflected sodium status. It was at the limits of detectionunder high sodium diet, but high under sodium restriction. Measuringangiotensinogen and related parameters in the urine should provideclinical indicators of the activity of this paracrine tubular RAS. Notonly should these correlate with sodium status, but they may also serveas markers of sodium sensitivity. Indeed, the hypothesis derived fromwork on A(−6) AGT mutation is that individuals homozygous for thisvariant would tend to maintain greater AGT expression under a highsodium diet than would individuals of other genotypes. This modestdifferential in the ability to down-regulate AGT under excess sodiumwould account for a relative propensity to retain more sodium, withlong-term attendant effects on blood pressure. This would account forsodium sensitivity in these individuals. Not only could A(−6) genotypeserve as a marker of this liability, as claimed earlier, but also, it ismore likely that urinary parameters reflecting the activity of thisnewly identified paracrine tubular RAS may prove of clinical value toidentify sodium sensitive individuals. These individuals would stand ahigher risk of developing essential hypertension when confronted withthe high sodium diet characteristic of affluent societies.

Certain parameters of the paracrine RAS can be measured in urine,specifically A-I, ANG, des-AI-ANG, and active renin (ANG denotesuncleaved, entire angiotensinogen protein, des-AI-ANG is the complementof the ANG protein after AI has been cleaved; as the peptide AI isexpected to be less stable and to degrade rapidly, ANG + des-AI-ANGreflect the total amount of ANG produced in proximal tubule). Theseparameters reflect an individual's sodium status, as ANG and renin aredown-regulated or up-regulated under high or low sodium, respectively.An individual can be classified as sodium sensitive if levels of AI,ANG, or des-AI-ANG are elevated under high salt diet compared to areference series of individuals. The parameters names above maycorrelate to genetic predisposition to essential hypertension measuredby AGT genotypes (M/T235 or A/G(−6)). These parameters may also be ofdiagnostic value in a number of clinical instances, including minimalrenal disease, diabetic nephropathy, IgA nephropathy, and disorderslikely to affect the function of the paracrine tubular RAS described inthis application.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and is not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1 Experimental Procedures

1. Generation of antibodies. Polyclonal antiserum was raised in rabbitsagainst highly purified mouse submaxillary gland renin according toMisono (50) and Geoghegan (51). 1:400-1:800 dilutions were used forimmunohistochemistry. The antiserum recognized submaxillary gland renin(ren-2) in crude salivary gland extracts and in purified fractions,ren-1 transiently expressed in COS-1 cells, as well as prorenin fromcrude kidney lysates and it did not cross-react with purified cathepsinD, total COS-1 lysates or crude liver extracts.

Polyclonal antiserum for mouse Ang was raised in rabbits against highlypurified mouse Ang purified as a glutathione-S-transferase (GST) fusionprotein (pGX-2T expression system, Pharmacia, New Jersey, N.Y.). Ang waspurified by glutathione affinity chromatography, GST-tag removal,anion-exchange, and gel permeation chromatography. 1:400 to 1:1200dilutions were used for immunohistochemistry.

2. In vitro Ang secretion studies. tsMPT were cultured as describedpreviously (19). tsMPT cells were grown on semi-porous membranes, mediawere concentrated by spin dialysis. Ang in cell medium or urine sampleswas measured as the release of A-I in a renin cleavage reaction (2.5 nMrenin in 25 mM NaOAc, pH 6.5, 0.5 mM AEBSF, 0.5 mM 8-hydroxyquinoline, 5mM EDTA). Generated A-I was measured with a competitive RIA (NEN DuPont,Boston, Mass.). Transepithelial resistance across the cell monolayer wasassayed using microelectrodes. Diffusion of tritiated mannitol from theapical into the basolateral chamber was used to measure integrity of thecellular monolayer. Blank filters and filters with non-continuous cellmonolayers served as controls. After subtraction of the electricalresistance of a blank filter, the transepithelial resistance values were70 Ω/cm² for an intact complete monolayer and 30 Ω/cm² for anon-continuous monolayer.

3. Animal experiments and measurements. C57BL6 mice were used followingIRB approved protocols. Twenty-four hours prior to dietary sodiummanipulations, all animals were fasted with free access to water,supplemented with 2% glucose and 0.1% KCl. Either one mg/kg amilorideand 2 mg/kg furosemide (52) or control carrier were applied bysubcutaneous injection. Low sodium (0.3% sodium) and high sodium (6%)diets were purchased from Purina Mills (Purina Mills Inc., St. Louis,Mo.). Blood was collected by cardiac puncture. Spot urine was collectedby bladder puncture. Hemidissected kidneys were formalin-fixed orsnap-frozen in liquid nitrogen. For urine collection, mice were placedin metabolic cages (Nalgene, Nalge Nunc International, Rochester, N.Y.).Urine specimens were collected at 12h intervals in tubes containingAEBSF and 8-hydroxyquinoline (NEN-DuPont, Boston, Mass.). Weight andurine volumes were recorded daily. Urinary Ang and A-I measurements werecorrected for creatinine or expressed as total urinary Ang. RNAisolation and RT-PCR were performed following standard protocols(QIAGEN, Valencia, Calif.). RT-PCR experiments were performed using theAccess RT-PCR system (Promega, Madison, Wis.).

4. In situ RT-PCR. In situ RT-PCR was performed as described by Ertseyand Scavo (53). Digoxigenin-labeled PCR product was detected in situusing an alkaline phosphatase conjugated anti-digoxigenin antibody(Roche, Indianapolis, Ind.) and visualized by adding the substratesnitroblue-tetrazolium and 5-bromo-4-chloro-3-indoyl-phosphate (NBT/BCIP;Sigma, St. Louis, Mo.). Sections were counterstained with PAS andphotographed.

5. Immunohistochemistry. Immunostaining was performed following standardprotocols (DAKO Co., Carpinteria, Calif.). The biotinylated secondaryantibody was detected using streptavidin conjugated with horseradishperoxidase or alkaline phosphatase and visualized with either3-amino-9-ethyl-carbazole (AEC; Sigma) or NBT/BCIP, respectively.Sections visualized with AEC were counterstained with hematoxylin andeosin.

6. Quantitative histology. Renin expression in renal tubular cells as afunction of sodium diet, or diuretics, was assayed by quantitating thefrequency of renin immunostaining in segments of the distal nephronusing the peroxidase reporter enzyme and AEC chromogen. Two independentblinded investigators scored sagittal kidney for renin using asubjective 0-4 scale: 0 equaled no tubular renin staining; 1 equaled atleast one positive cell per tubule segment; 2 equaled between 25-50% ofcells per of tubule segment; 3 equaled more than 50% of cells per tubulesegment stained for renin; 4 equaled>75%. Concordance was reproduciblygreater than 90%. Four separate experiments were performed. Results wereanalyzed by comparing the mean±S.E.M. between groups using unpairedt-tests. p<0.05 was considered significant.

7. Cell Immunoblotting. Arcades of connecting tubules weremicrodissected following limited collagenase digestion and isolated. Thepurity of the isolated junctions was checked by microscopy.Isolatedjunctions were further collagenase digested to obtain singlecells (0.5% at 37° C. for 10 min). Cells were washed and resuspended in30 μl low sodium containing tissue culture medium (serum free DMEM),dropped on a PVDF membranes (MultiScreen-IP, 0.45 μm Hydrophobic, HighProtein Binding Immobilon-P membrane Millipore Brdford, Mass.) andincubated overnight. CHO cells transfected with mouse renin and humanAng served as positive and negative control. Immunoblotting wasperformed as described previously (22-24). Following overnightincubation, cells were fixed on the membrane using 4% paraformaldehyde.Anti-mouse renin antibody, biotinylated anti-mouse IgG (DAKOCorporation, Carpenteria, Calif.), and streptavidinealkaline-phosphatase (DAKO) were used at 1:500 dilutions.AIkaline-phosphatase was detected using the NBT/BCIP (Sigma) chromogen.Membranes were mounted and photographed.

Example 2 Proximal Tubule Epithelium Secretes Ang at its Apical Side

Angiotensinogen expression in whole kidney tissue was examined byimmunohistochemistry in sodium restricted animals. Staining was observedonly in proximal tubules, and the granular appearance of the protein wasin the vicinity of their PAS-counterstained brush-border rich luminalmembranes suggesting a secretory process (FIG. 1A). To test thishypothesis, confluent monolayers of conditionally immortalized cells ofmurine proximal tubule (19) were grown on semipermeable membranes, whichseparated apical and basolateral compartments. The integrity of themonolayers was established by visual inspection, by demonstration of asignificant transepithelial resistance, and by monitoring the diffusionof tritiated mannitol placed in the apical chamber. Using intactmonolayers, Ang was reproducibly detected in the apical but not in thebasolateral compartment (FIG. 1B). Under our prevailing experimentalconditions, AGT mRNA was detected by Northern blot of total RNA (19)while renin mRNA was too close to the detection limits of RT-PCR to beconclusive.

If Ang is secreted in tubular lumen, is it present in final urine?Indeed, the protein was detected in the urine of mice and men by WesternBlot analysis with specific polyclonal antiserum. Native Ang wasmeasured in 12 h-urine of male mice kept in metabolic cages withunrestricted access to food and water, conditions that did notsignificantly affect body weight and therefore total body water. UrinaryAng was inversely related to dietary sodium (FIG. 1C). Native Ang wasalso observed in urine specimens of healthy human volunteers atconcentrations ranging from 66±7 to 523±33 pM (FIG. 1D).

Example 3 Renin Is Synthesized by Principal Cells of Connecting Tubule

Transit of Ang through the entire nephron reflects either elimination,delivery to a downstream site of renin expression, or both. To addressthis issue, the distribution of renin in the kidney was examined byimmunohistochemistry with an antiserum raised against purifiedsubmaxillary gland renin (ren-2). As expected, intense staining wasobserved in JGA (FIG. 2B, arrow). In sodium restricted animals, stainingwas also observed unambiguously in the mid-cortical arcades formed byconnecting segments of mid cortical and deep nephrons, but not in othersegments (FIGS. 2A, B). The specificity of renin staining was confirmedby several observations. Staining was absent in sections treated withpreimmune rabbit serum or after preincubation with antigen (FIG. 2D).Furthermore, our observations were confirmed using a previouslyestablished polyclonal renin antiserum (20). For each antiserum, reninimmunoreactivity was jointly observed in JGA and cortical segments ofdistal nephron over the entire dilution series tested. Immunoreactiverenin was also detected in similar segments of human kidneys usinganti-human renin antiserum (FIG. 2E) (20).

Segments of the nephron distal to the macula densa can be subdividedinto distinct entities on the basis of anatomical and functionalfeatures. Contrary to proximal segments, distal segments exhibitsignificant cellular heterogeneity. In addition to intercalated cell,distal segments have variable numbers of principal cells with featuresand functions that vary among segments. Cortical distal segments includethe distal convoluted tubule (DCT), connecting tubule (CNT), andcortical collecting duct (CCD). The topographical distribution oftubular renin immunostaining supports the conclusion that it is presentmainly in CNT segments on the basis of the following arguments: (1)staining is not observed in the larger cortical collecting ducts; (2)cellular staining is observed in cross- and longitudinal- sections oftubules in the cortical labyrinth located in the immediate vicinity ofcortical radial veins (FIGS. 1A, B); (3) these clusters are onlyobserved in the midcortical labyrinth demarcated by medullary rays. Thistopographical arrangement is characteristic of the arcades formed bymerging connecting tubules of midcortical and deep nephrons (21).

The epithelium of connecting tubules is composed of two main cell types.The intercalated cells (IC) are subdivided into two subtypes, α and β;both express H⁺-ATPase, whereas only the β subtype stains for peanutlectin. Staining of serial sections for renin, H⁺-ATPase (FIGS. 2F, G)or peanut lectin revealed that cells staining for renin did not stainfor H⁺-ATPase or peanut lectin, suggesting that they are not IC. Themorphology of renin-positive cells is consistent with that reported forcortical connecting tubule (CNT) cells, with a characteristic polygonalappearance, with a convex apical side devoid of brush border, and acentrally located nucleus within an abundant clear cytoplasm (21).Notably, renin staining predominates in the apical segment of thecytoplasm and in the vicinity of the nucleus.

The hypothesis of local renin synthesis, as opposed to uptake of reninof systemic or proximal tubular origin, was tested by a combination ofmicrodissection of connecting tubule arcade and RT-PCR. Duringmicrodissection, glomeruli (FIGS. 3A, C), proximal convoluted tubules(FIGS. 3A, B) loops of Henle and blood vessels were readily identifiableand separated from arcade junctions between connecting tubules andeither connecting tubules or cortical collecting ducts (FIGS. 3A, D).Renin amplification products of expected size and sequence were clearlyobserved in RNA preparations from glomerulus independently of dietarysodium (FIG. 3E). Unambiguous signal was also observed in isolatedconnecting tubule arcades particularly in sodium restricted animals.Only minimal signal was noted in proximal tubule under sodiumrestriction. These observations were reproduced in four independentseries of microdissection experiments. Controls included amplificationof GAPDH for RNA quality and α-smooth muscle actin to excludecontamination of tubular segments with JGA components. The specificityof all amplification products was confirmed by DNA sequencing.

To confirm renin synthesis in connecting tubules of the nephron by anindependent method, in situ RT-PCR was applied to kidney sections frommice subjected to 16-hour sodium restriction (FIGS. 3F-K). Renin mRNAwas unambiguously identified in cells of cortical segments of the distalnephron and in cells of afferent arterioles (FIGS. 3I-K). Renin mRNA wasnot detected in proximal tubules (FIGS. 3I, J arrowhead). Nor was itdetected in the inner or outer medulla. Control sections not pretreatedwith DNase showed uniform staining of all cells in all nephron segments(FIGS. 3F, G); control sections, DNase treated but not reversetranscribed, showed no staining (FIG. 3H). Further evidence ofspecificity was provided by the absence of signal when primers wereapplied to samples that were not reverse transcribed. The specificity ofthe primers used for amplification was validated by DNA sequencing incontrol RT-PCR experiments. To further ensure that amplification wasspecific and not the result of primer extension of fragmented genomicDNA, control amplifications were performed from reaction supernatants.

Example 4 CNT Cells Secrete Renin

Renin secretion by CNT cells was demonstrated using cell immunoblotting(22-24). Isolated cells from microdissected arcades of connectingtubules secreted renin (FIG. 3L). CHO cells expressing mouse renin (FIG.3; M) and human angiotensinogen (FIG. 3; N) served as positive andnegative control respectively. Renin secretion was revealed bypericellular halos of immunoreactive renin.

Example 5 Renin Expression in CNT Varies with Dietary Sodium

On the basis of immunostaining and expression studies, we conclude that,in addition to its major site of expression in JGA, renin is alsoexpressed in connecting tubule. In subsequent studies, we have usedrenin immunostaining to investigate the relationship between dietarysodium and CNT renin after overnight manipulation of tubular sodiumdelivery by varying total sodium intake and/or sodium reabsorption atspecific sites through diuretics. Furosemide inhibits the Na⁺/K⁺2Cl⁻transporter upstream of the distal tubule, whereas amiloride, a sodiumchannel blocker, affects sodium reabsorption in distal segments of thenephron. Because of signal saturation of renin immunostaining in JGA,renin expression at this site was estimated by semiquantitative RT-PCRof total kidney RNA (25). Renin expression in CNT cells was assessed byquantitative histology (frequency of CNT cells staining for renin).Scoring was performed by two independent, blinded observers in fourindependent sets of replicate experiments. Under high sodium, animalsexhibited minimal renin staining in CNT and moderate JGA reninexpression (FIG. 4A; FIGS. 4D, E group 1A). By contrast, the combinationof high sodium and amiloride administration led to a marked increase inCNT immunoreactive renin (FIG. 4B; FIGS. 4D, E group 1B); JGA renin wassignificantly decreased (p<0.05). Overnight sodium restriction led to amarked increase in CNT immunoreactive renin. (FIG. 4C, FIGS. 4D, E group2A) but no significant change in JGA renin. However, longer periods ofsodium restriction stimulated renin expression in JGA. The combinationof sodium restriction and furosemide resulted in decreased reninexpression in CNT, without additional effects on JGA renin (FIGS. 4D, Egroup 2B). Manipulation of dietary sodium was monitored by measuringtotal sodium excretion. Under these experimental conditions, thetreatments were without effect on body weight, therefore excludingsignificant variation in total body water.

The observations of the present invention support the followingconclusions: (1) angiotensinogen is synthesized by proximal tubule andsecreted into tubular fluid; (2) uncleaved Ang transits through theentire nephron and can be found in final urine; (3) renin is synthesizedand secreted by CNT cells and (4) both proximal angiotensinogen anddistal renin expression vary as a function of dietary sodium. Togetherwith filtered renin, the spatial distribution of these elements of atubular RAS and their correlation with dietary salt suggest that theyplay an important role in the coordinated regulation of sodiumreabsorption at various sites within the nephron.

The presence of AGT mRNA in proximal tubule and its variation withsodium intake has been observed previously in whole kidney sections (5).Besides confirming these findings, the data presented hereincharacterize the time-course of AGT expression in parallel with that ofrenin in distal segments of the nephron and with the urinary excretionof Ang and A-I. Furthermore, they demonstrate luminal secretion of Angby polarized epithelium; previous experiments with primary culture ofheterogeneous cell populations from kidney cortex suggested secretion ofthe protein but could not resolve the directionality of this process(26). The observations concerning urinary Ang confirm prior reportsusing laboratory animals (27-29). In the past, urinary angiotensinogenwas thought to be a clinical indicator of damage to the glomerularmembrane, since Ang is normally not filtered (30, 31).

Secretion of Ang to the apical side of cultured monolayers does not initself provide definitive evidence for luminal secretion of the proteinby proximal tubule in vivo. The apical distribution of Ang secretorygranules in proximal tubule (FIG. 1A) suggests such a process. So doesthe presence of Ang in final urine in a direct correlation with AGTexpression in proximal tubule and in an inverse relationship withdietary sodium. Taken together, these observations strongly suggest thatAng is indeed secreted in tubular fluid in this initial nephron segment.If so, filtered renin of systemic origin could act on luminal Ang togenerate A-II. The functional significance of A-II as a major regulatorof sodium transport in this segment, in part through its stimulation ofthe sodium-hydrogen exchanger, NHE-3 (9, 10, 32), is well documented(4). With this hypothesis, ultrafiltration of systemic renin would notbe regarded only as an elimination route for the enzyme, as it wouldalso serve an important function in regulating bulk sodium reabsorptionin proximal tubule

Previous reports have suggested the existence of an autocrine RAS inproximal tubule, primarily on the basis of the detection of renin mRNAby RT-PCR of total RNA from selected cell populations or frommicrodissected segments of proximal tubule (8, 33-35). The presentobservation of intracellular A-I and A-II formation in tsMPT and faintrenin amplification product in microdissected segments of proximaltubule in sodium restricted animals are consistent with this model. Theexpression levels of renin and angiotensinogen in proximal tubule aremarkedly different, however. Angiotensinogen protein appears abundant inepithelium of proximal tubule, whereas renin is below the detectionlevel of immunohistochemistry when antibodies are used at dilutionsensuring specificity. Likewise, AGT mRNA is detected by Northern blot oftotal RNA. By contrast, evidence for a renin transcript at this siteescapes even in situ hybridization after RT-PCR, a faint signalappearing only in liquid-phase RT-PCR. The functional significance ofthe latter observation remains unclear, as faint RT-PCR signals canreflect either legitimate or illegitimate transcription. Whereas thereaction between filtered renin and secreted Ang may be the predominantmechanism of formation of A-II in tubular fluid of the proximal tubule,an autocrine or intracrine local RAS, expressed at a much lower level,may still serve a distinct purpose in the homeostasis of this nephronsegment.

Renin immunoreactivity has been occasionally noted in tubular segmentsof mouse kidney (36-39). In one case, where the focus of theinvestigation was on JGA renin, it was dismissed as an experimentalartifact (38). In other instances, the authors relied on indirectarguments to suggest that it represented non-specific uptake of filteredrenin. In the present work, the hypothesis of local synthesis wasexamined directly following two distinct experimental approaches.Concordant results obtained in repeated series of four independentexperiments for each of two different methods strongly support thehypothesis of local renin synthesis. The predominantly apicaldistribution of renin immunostaining in tissue sections (FIG. 2C) andthe demonstration of renin secretion by isolated CNT cell in vitrosuggests that renin is secreted into tubular lumen. The observation ofrenin in final urine alone does not settle this issue, however, as itcannot be excluded that some of the filtered renin of systemic originescapes degradation in proximal tubule. Taken together, these datasuggest that renin secreted by connecting tubule could act on luminalangiotensinogen that originated in proximal tubule to release A-I inluminal fluid. The documented presence of both ACE and A-II receptors incollecting duct (15, 16) would allow formation and action of A-II indistal segments of the nephron. Because of poor accessibility to directexperimental investigation, little is known about the effect of A-II inconnecting tubule and collecting duct. One report does suggest thatluminal A-II stimulates amiloride-sensitive sodium transport in theinitial collecting tubule of cortical nephrons (11).

The distribution of renin immunostaining in connecting tubule arcades isstrikingly similar to, the site of expression of tissue kallikrein inkidney. While colocalization of renin and kallikrein remains to beestablished, it has indeed been shown that tissue kallikrein secretedinto distal tubule (40) originate in CNT (41), with predominantimmunostaining at the apical side of CNT cells in a pattern quitesimilar to that observed here for renin (42, 43). It is also known thatkininogen is synthesized and secreted in tubular lumen by principalcells of the collecting duct, and bradykinin B2 receptors have beenreported at the luminal side of this nephron segment (44). The presenceof components of both the renin-angiotensin and the kallikrein-kininsystems in the luminal compartment of connecting tubule and collectingduct suggests that the two systems may play a coordinated, balanced rolein the fine regulation of the concentrations of sodium and potassium infinal urine. These systems are interrelated not only through the oftendescribed opposite actions of their effectors, A-II and bradykinin, butalso through multiple areas of potential overlap, such as aldosteroneresponse, sodium and potassium balance, renin activation, and peptideconversion through the action of ACE. Connecting tubule would appear tobe a very strategic site in this coordinated regulation.

The experimental work of Guyton and his colleagues has long establishedthe significance of the pressure-natriuresis relationship in theregulation of baseline blood pressure, and the dominant role ofintrarenal A-II in the regulation of sodium balance in response tovariation in dietary sodium (45, 46). The genetics of rare mendelianhypertension such as Liddle syndrome (47) or the syndrome ofmineralocorticoid excess (48, 49) confirm experimental physiology bystressing the significance of sodium reabsorption in distal segments ofthe nephron in blood pressure regulation. Angiotensinogen of proximaltubular origin and renin expressed by connecting tubule may provide amechanism to coordinate the functions of proximal and distal segments ofthe nephron in regulation of sodium balance and blood volumehomeostasis. It may be through this system that molecular variation atthe AGT locus (18) affects individual liability to develop essentialhypertension.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

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What is claimed is:
 1. A method for determining the sensitivity of anindividual to sodium, wherein sodium sensitivity is defined as anindividual's tendency to respond to excess sodium intake with anincrease in blood pressure, said method comprises measuring the amountof a substance selected from the group consisting of angiotensinogen,angiotensin-I and des-angiotensin-I-angiotensinogen in the urine of anindividual under normal physiological conditions except said individualis on a high salt diet and comparing said amount with a referencestandard, wherein an elevated amount of said substance in an individualon a high salt diet under normal physiological conditions is indicativeof sodium sensitivity.
 2. A method for assessing the function of therenal proximal tubule in an individual, which comprises measuring theamount of a substance selected from the group consisting ofangiotensinogen, angiotensin-I and des-angiotensin-I-angiotensinogen andrenin in the urine of an individual under normal physiologicalconditions and said individual is not on a high salt diet and comparingsaid amount with a reference standard, wherein an elevated amount ofsaid substance in an individual under normal physiological conditions isindicative of a higher tubular flow and a reduced amount of saidsubstance in an individual under normal physiological conditions isindicative of a lower tubular flow.